1. Field of the Invention
This invention relates to the stabilization of labile coenzymes in liquid media.
2. Description of the Prior Art
It has recently been estimated that 25% of all in vitro diagnostic tests conducted annually in this country are not reliable. Unreliable tests can result in unnecessary medical treatment, the withholding of necessary treatment and lost income. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement stems from the fact that the exact nature of enzymes, as well as the mechanisms of their action, remain unknown for the most part. At present, the greatest limitation on the enzyme reagent manufacturer, by far, lies in the unstable characteristics of his products. Current methodologies require the use of numerous labile ingredients, and these ingredients are more likely to increase, rather than decrease, in number.
The present commercial state of the art used for stabilizing the reactive ability of enzymes or coenzymes is by locking them into a solid matrix either by freeze drying, dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and usefulness of the product is further limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve. This condition is exemplified by the fact that most commercial freeze dried control sera (reference serum) list the acceptable bottle to bottle variation of enzyme constituents at .+-.10% of the mean.
In the clinical diagnostic field the commercial application is represented by, but not limited to, the diagnostic reagents used to determine and quantitate the following constituents in biological fluids:
1. Glutamic-oxalacetic transaminase (SGOT); PA0 2. glutamic-pyruvic transaminase (SGPT); PA0 3. lactic dehydrogenase (LDH); PA0 4. creatine Phosphokinase (CPK); PA0 5. .alpha.-hydroxybuteric dehydrogenase (.alpha.-HBD) PA0 6. glucose (via Hexokinase-G-6-PDH). These reagents react similarily, contain some common labile ingredients, and some of the chemical reactions involved are common. The following chemical reaction scheme is presented as a model to illustrate the general nature of the reactions involved: ##STR1##
All enzymatic reactions listed above will follow this general scheme, where reaction (2.) is usually referred to as the coupling reaction, reactions (2.) or (3.) are the measuring reactions, and reaction (1.) may be characterized as the primary reaction. It is understood however, that not all three reactions are required for measurement; in fact, they may be limited to two, or one. In the case of the ultraviolet measurement of lactic dehydrogenase (LD) activity, only reaction (2.) is involved, as follows: